The signal recognition particle (SRP) binds the N-terminal polypeptide signal sequence emerging from a translating ribosome and targets this ribosomal complex to a receptor at the endoplasmic reticulum membrane. Polypeptides are translocated into the intermembrane space in a GTP-regulated manner. The mammalian SRP is comprised of two domains: the Alu and large subunits. The large subunit (LS) contains one-half of the RNA (-150 nts) and the SRP19, SRP54, and SRP68/SRP72 heterodimer proteins. The large subunit performs most of the functions of the SRP including signal sequence recognition, GTP binding and hydrolysis, receptor docking, and, in part, elongation arrest. The long term goals of this project are to understand, in a fundamental and quantitative way, the interplay of protein and RNA components and of binding by signal peptide and nucleotide substrates in the cooperative assembly and functioning of the large subunit of the mammalian SRP. Specific aims are: (1) To determine the molecular basis for the strong cooperativity in the assembly and function of SRP19 and SRP54. (2) To understand the mechanism of assembly of the natively unfolded SRP19 protein with the SRP RNA. (3) To map at nucleotide resolution the RNA binding sites for the individual SRP68 and SRP72 proteins and for the SRP68/72 heterodimer and to quantify cooperative binding with SRP54 and SRP19. Rigorous analysis of these experiments is made possible by the development, in our lab, of robust single-nucleotide resolution approaches for determining equilibrium binding constants in multi-component RNA-protein complexes. (4) To analyze interaction of the complete SRP large subunit with its two classes of substrates: guanosine nucleotides and signal peptides. Overall, this work will address key features of how cooperative interactions between SRP protein and RNA components function to carry out signal peptide recognition and nucleotide binding. Moreover, the work is designed to identify principles generalizable to other biologically and medically prominent ribonucleoprotein complexes.